Contamination of for example malt alcoholic beverages by harmful microorganisms is undesirable as it causes turbidity, off-flavor, or other adverse effects, which deteriorates product quality. Specifically, Lactobacillus, Pediococcus, and Pectinatus are some of the representative examples of harmful bacteria in beer. Among these bacteria, Lactobacillus brevis and Lactobacillus lindneri are particularly well known as beer spoilage bacteria. In a method most commonly used these days to detect such harmful bacteria, a sample of beer to be tested is filtered with a membrane filter and cultured in an appropriate medium to detect resulting colonies.
In one method of determining the identity of the colony-forming bacteria as being harmful, PCR is carried out with primers specific to the bacteria, using the extracted DNA of the colony-forming bacteria as a template, and the presence or absence of a PCR product is determined by electrophoresis (see, for example, Japanese Laid-Open Patent Publication No. 289295/1995 (Tokukaihei 7-289295; published on Nov. 7, 1995)). However, the method requires multiple rounds of PCR reactions, requiring a large number of primers for each different species of bacteria. The method therefore requires complicated procedures, and is time consuming. For shorter reaction time, the primers may be mixed together to carry out amplification in a single tube. However, since there is a limit in the number of primers that can be used, it is difficult to obtain accurate results based on a trace amount of specimen.
In order to solve these problems, there have been proposed different methods of identifying bacterial species. In these methods, primers are designed in such a manner that only a portion of a gene that commonly exists across different bacterial species and that contains species-specific sequences is amplified. After the amplification, the amplified product is excised by restriction enzymes that recognize the species-specific sequences, and bacterial species of the gene are identified based on the band size obtained by electrophoresis. However, it is not necessarily the case that such restriction enzymes are available for all species of bacteria to be tested. Further, since the methods require the highly complicated procedure of excising the amplified product with several different kinds of restriction enzymes for electrophoresis, the methods are also problematic in terms of speed and convenience.
These problems can be solved by methods employing hybridization, in which a species-specific sequence is used as a probe, and the presence or absence of a complementary sequence in the tested DNA or RNA is determined by hybridization.
However, the methods employing hybridization also pose a problem in that crosshybridization with other species often occurs between closely related species having a high level of DNA homology. As this is often the case, it is difficult to accurately identify bacterial species.
The methods having the problem of crosshybridization are particularly problematic when used for Lactobacillus bacteria, one of representative examples of beer spoilage bacteria, because many species of these bacteria are very closely related to one another. As such is the case, the methods cannot be used for the detection and identification of harmful bacteria.